PROJECT SUMMARY Immune related adverse events (IRAEs) remain a major obstacle to optimal application of combination checkpoint blockade in human cancer. Deeper understanding of the mechanisms underlying these IRAEs, and in particular underlying cellular and genomic pathways and antigenic targets is essential to develop optimal strategies to prevent and treat these AEs. This application represents a collaborative effort between two investigators with experience in cancer immunology (KD) and human autoimmunity (IS) to investigate the role of B cells in IRAEs. It builds directly on our prior studies evaluating early immunologic changes in patients undergoing immune checkpoint blockade, as well studies of altered B cell differentiation and function underlying human autoimmune disorders. In these studies, we have observed that CCB therapy leads to a distinct pattern of changes in B cells, particularly involving CD21lo B cell subset and plasmablasts. We have also identified distinct epigenetic and transcriptional signatures of B cells in autoimmune disorders such as lupus. Our hypothesis is that preexisting changes in autoreactive B cells will identify patients at risk for early IRAEs. We further posit that CCB-induced expansion of pathogenic B cells is mediated by the engagement of epigenetic and transcriptomic programs akin to those observed in naturally occurring autoimmunity. B cells have been proposed to play both a pro- and anti-tumor role in cancer. In order to specifically isolate the effect of B cells in IRAEs, we will evaluate changes in pathogenic B cells and plasmablasts in the context of a randomized clinical trial to specifically deplete B cells in melanoma patients undergoing CCB. The specific aims of the project are: Aim 1. To assess whether pre-existing transcriptional and epigenetic alterations in B cell subsets identify patients at risk for CCB-induced irAEs and are normalized by Rituxan Aim 2. To evaluate Rituxan-mediated depletion of pathogenic B cells and their contribution to irAEs following CCB. Aim 3. To evaluate whether depletion of B cells leads to alterations in circulating or tumor-infiltrating T cells in patients undergoing CCB. Together these studies will provide direct insights into the relative contribution of B cells in patients undergoing CCB, both in terms of favorable as well as adverse impact on tumor-immune interactions and patient outcome. Understanding these cellular interactions directly in humans is essential for developing rational approaches to prevent IRAEs.